Host factors are key determinants of RNA virus pathology, host - virus interactions, and evolution of viruses. Recent genome-wide screens with several viruses have identified several host proteins, which directly inhibit +RNA virus accumulation. These host proteins might be novel components of the host innate systems against viruses. Among the identified inhibitory host proteins are the family of cyclophilins, which are effective against several viruses. Cyclophilins are a large family of ubiquitous, highly conserved proteins that have peptidyl-prolyl isomerase and protein chaperone activities. Cyclophilins are involved in several diseases, such as human cancers, neurodegenerative diseases and viral infections. Determining the mechanism of inhibition by host factors, like cyclophilins, is a major frontier in current virology research. The identified inhibitory host factors could be used in antiviral approaches with several advantages over traditional antivirals, including broader antiviral effects against many related viruses and more durable antiviral effects since viruses may have a more difficult challenge to evolve mutants that evade these host factors. Progress in our understanding of the mechanisms of the inhibitory activity of host factors is slow due to functional redundancy in the host cells or lack of knowledge about their functions. However, easily tractable virus - host systems, such as Tomato bushy stunt virus (TBSV) and yeast as a model host can greatly contribute to our understanding of the functions of these host proteins. This project will likely advance our understanding of the role of cyclophilins in virus-host interactions. This advance could immensely help other scientists working with less tractable, but devastating viral pathogens for which similar studies are currently not yet feasible. The gained knowledge will not only be useful for TBSV, but to other significant human pathogens, such as West Nile virus, Dengue virus and other flaviviruses and pestiviruses, due to the similarity of their replicase proteins. Collectively, the major advances with TBSV will stimulate development of new approaches for studying RNA replication and host - pathogen interactions for important human pathogens. The following are the major strengths of the proposal: (i) Viral RNA replication is clearly of immense importance for viruses to infect living organisms. (ii) Cyclophilins are major proteins affecting viral and other human diseases, yet their functional roles are currently poorly defined. (iii) The combination of yeast and authentic cell-free assay developed by the investigator is currently the most potent for studying the mechanism of host factors involvement in viral RNA replication and viral pathogenesis. (iv) The research holds promise of benefiting society by leading to groundbreaking results in the area of virus replication, host-virus interactions and the adaptation of viruses to their hosts.